Apoptosis inducer for cancer cell

ABSTRACT

The present invention revealed that by suppressing the expression of the WRN gene, the BLM gene, or the RecQ1 gene, which belong to the RecQ helicase family, apoptosis is induced in various cancer cells and their proliferation is suppressed. Compounds that suppress the expression of RecQ helicase family genes or the functions of RecQ helicase proteins are thought to have the activity of inducing apoptosis.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing 390081_(—)401D1_SEQUENCE_LISTING.txt. The text fileis 163 KB, was created on Sep. 27, 2012, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to apoptosis inducers for cancer cells,which comprise as an active ingredient a compound that suppresses theexpression of a gene belonging to the RecQ DNA helicase family, or acompound that suppresses the function of the protein encoded by thatgene, and screening methods to select candidate compounds for theapoptosis inducers.

BACKGROUND ART

Genes belonging to the RecQ DNA helicase family are widely present inorganisms ranging from prokaryotes such as Escherichia coli (E. coli) tohigher eukaryotes including humans. Conserved in the evolution process,these genes diversified along with the multicellularization oforganisms. The E. coli RecQ gene was the first of the RecQ family genesto be discovered. This gene was identified as a gene participating inzygotic recombination and in the RecF pathway for UV damage repair (seeNon-Patent Document 1). E. coli RecQ gene has been revealed to have thefunction of suppressing incorrect recombinations (see Non-PatentDocument 2). The budding yeast SGS1 gene and the fission yeast Rqh1 geneare the only known RecQ homologues in these yeasts. Both of these genesmainly suppress recombination and play important roles in genomestabilization (see Non-Patent Documents 3 and 4). Higher eukaryotescarry a plurality of RecQ homologues. In humans, there are five types ofgenes known to belong to the RecQ family: the RecQL1 (see Non-PatentDocument 6), BLM, WRN, RTS, and RecQL5 genes. Of these five, the RTSgene (see Non-Patent Document 5, and Patent Documents 1 and 2) and theRecQL5 gene (see Non-Patent Document 5, and Patent Document 3) wereidentified by the present inventors. BLM, WRN, and RTS genesrespectively cause Bloom's syndrome (see Non-Patent Document 7),Werner's syndrome (see Non-Patent Document 8), and Rothmund-Thomsonsyndrome (see Non-Patent Document 9). These genes all play importantroles in genome stabilization in cells.

In fibroblast cells and lymphocytic cell lines derived from patientswith Werner's syndrome, chromosomal translocation and deletion, whichare indexes for genome instability, have been reported to occur with ahigh frequency (see Non-Patent Document 10). Chromosomal breakage andsister chromatid exchange (SCE) are frequently detected in cells derivedfrom patients with Bloom's syndrome (see Non-Patent Document 11).Trisomies of human chromosome 2 and 8 are frequently found inlymphocytes derived from patients with Rothmund-Thomson syndrome (seeNon-Patent Document 12). These findings suggest that WRN helicase, BLMhelicase, and RTS helicase encoded by the various causative genes ofthese three genetic diseases play important roles in genomestabilization in cells.

Telomere length abnormalities are seen in lymphocytic cell lines derivedfrom patients with Werner's syndrome compared to cell lines derived fromnormal healthy subjects (see Non-Patent Document 13). In addition, cellimmortalization was not observed in lymphocytic cell lines derived frompatients with Werner's syndrome, although about 15% of cell linesderived from normal healthy subjects were immortalized after passaging(see Non-Patent Document 14). This finding indicates that WRN helicasecontributes to telomere structure maintenance, and is thus essential forthe immortalization (canceration) of lymphocytic cell lines.

It has been suggested that WRN helicase is associated with homologousrecombination-mediated repair, because the helicase forms foci in thenucleus in response to DNA-damaging agents, and these foci areco-localized with the single-stranded DNA-binding protein RPA (which isa WRN-binding protein) and with the recombination repair factor RAD51(see Non-Patent Document 15). In addition, WRN helicase has been knownto bind to the DNA-dependent protein kinase complex (DNA-PK) and to flapendonuclease 1 (FEN-1). By binding to DNA-PK, WRN helicase plays animportant role in the processing of terminals generated by DNA doublestrand breaks, which are repaired in the pathway of non-homologous endjoining (see Non-Patent Document 16). WRN helicase is believed toactivate FEN-1 by binding to it, and to provide a site for precisereconstruction of the replication fork through homologous recombinationby processing Okazaki fragments (see Non-Patent Document 17). The abovefindings suggest that WRN helicase plays an important role in DNA repairduring DNA replication.

BLM helicase is localized in the PML body, a specific structure found inthe nucleus, and it binds to topoisomerase III (see Non-Patent Document18). The helicase has the unwinding activity of the G-quadruplexstructure, and thus is considered to contribute to telomere maintenance(see Non-Patent Document 19). Furthermore, the helicase has beenreported to unwind the Holliday junction and to interact with the Rad51protein (see Non-Patent Document 20). These findings suggest that BLMhelicase cooperates with other DNA-metabolizing enzymes and playsimportant roles in recombinational DNA repair and telomere maintenance.

Of the five human proteins belonging to the RecQ DNA helicase family(RecQ1, BLM, WRN, RTS, and RecQ5), RecQ1, BLM, WRN, and RTS areexpressed at negligible levels in resting cells, whereas they areexpressed at high levels in cells whose proliferation has been enhancedby transformation with viruses (see Non-Patent Document 21).Furthermore, when the carcinogenic promoter TPA is added to restingcells, the expression of RecQ1, BLM, WRN, and RTS is induced along withthe induction of cell division (see Non-Patent Document 21). Thesefindings suggest the importance of the RecQ DNA helicase family in cellproliferation.

Taken collectively, these findings suggest that the RecQ DNA helicasefamily members may be potential target molecules for anti-cancer therapybecause the family members participate in genomic repair in cells (BLM,WRN and RTS) and also the maintenance of telomere structure (BLM andWRN), play important roles in the immortalization of certain cells(WRN), and their expression is induced following cell division (RecQ1,BLM, WRN and RTS).

In addition, the expression levels of genes in the RecQ DNA helicasefamily are markedly higher in tumor cells. Thus compounds that suppresstumor growth can be screened using the suppression of expression of RecQDNA helicase family genes as an index (see Patent Document 4). It hasalso been suggested that compounds suppressing RecQ helicase geneexpression may suppress cancer cell growth (see Patent Document 4).

However, no one has reported the correlation between the suppression ofRecQ DNA helicase family gene expression and the cancer cell-specificinduction of apoptosis, and there have been no findings that suggestsuch a correlation.

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DISCLOSURE OF THE INVENTION

The present invention was achieved under such circumstances. Anobjective of the present invention is to provide apoptosis inducers forcancer cells, which comprise as an active ingredient a compound thatsuppresses the expression of a RecQ DNA helicase family gene, or acompound that suppress the function of the protein encoded by that gene.Another objective is to provide screening methods to select candidatecompounds for apoptosis inducers for cancer cells.

The present inventors conducted extensive research to achieve theabove-described objectives. The expression levels of RecQ DNA helicasefamily genes are known to be higher in tumor cell lines (for example,cancer cells). Using siRNAs having the activity of suppressing humanRecQ helicase family gene expression, the present inventors studied theinfluence of suppressing the expression of respective RecQ helicasefamily genes on cancer cell growth. As a result, the present inventorsfound, for the first time, that apoptosis can be induced in variouscancer cells by suppressing the expression of the RecQ helicase familygenes WRN, BLM, or RecQ1, and that cancer cell growth can thus besuppressed through this apoptosis. These effects were not observed in adiploid fibroblast cell line derived from normal human fetal lung. Inaddition, the present inventors found that, without significant sideeffects, WRN-siRNA and RecQ1-siRNA can inhibit in vivo tumor growth incancer-bearing nude mice.

Thus, the RecQ helicase family genes WRN, BLM, and RecQ1 can serve astarget molecules for superior anticancer agents having few side effects.Specifically, the above-described compounds are able to suppress theexpression of RecQ helicase family genes, or the function of RecQhelicase proteins, and may have the activity of inducing apoptosis. Suchcompounds are expected to serve as anticancer agents having few sideeffects. For example, compounds based on the mRNA sequences of WRN, BLM,and RecQ1 that suppress the expression or activity of RecQ helicasefamily proteins such as siRNA, are expected to serve as anticanceragents having few side effects.

Apoptosis inducers can be screened by using the expression of RecQhelicase family genes as an index.

Specifically, the present invention relates to apoptosis inducers forcancer cells which comprise as an active ingredient a compound thatsuppresses the expression of a RecQ DNA helicase family gene, or acompound that suppresses the function of the protein encoded by thatgene, and screening methods to select candidate compounds for apoptosisinducers for cancer cells. More specifically, the present inventionprovides the following:

[1] an (cancer cell-specific) apoptosis inducer for cancer cells,wherein the apoptosis inducer comprises as an active ingredient acompound that suppresses the expression of a gene belonging to the RecQDNA helicase family;

[2] the apoptosis inducer according to [1], wherein the compound thatsuppresses the expression of a gene belonging to the RecQ DNA helicasefamily is a double-stranded RNA having RNAi activity towards that RecQDNA helicase family gene;

[3] the apoptosis inducer according to [2], wherein the double-strandedRNA comprises a sense RNA comprising a sequence homologous to anarbitrary 20 to 30 consecutive nucleotides from the mRNA of a genebelonging to the RecQ helicase family, and an antisense RNA, comprisingthe sequence complementary to the sense RNA;

[4] an apoptosis inducer for cancer cells, wherein the apoptosis inducercomprises as an active ingredient a DNA that can express thedouble-stranded RNA having RNAi activity towards a RecQ DNA helicasefamily gene;

[5] the apoptosis inducer according to [1], wherein the compound thatsuppresses the expression of a RecQ DNA helicase family gene is either:

-   (a) an antisense nucleic acid targeting a transcript of the gene    belonging to the RecQ DNA helicase family, or a portion thereof; or-   (b) a nucleic acid having the ribozyme activity of specifically    cleaving a transcript of the RecQ DNA helicase family gene;

[6] an apoptosis inducer for cancer cells, which comprises as an activeingredient a compound that suppresses a function of a protein encoded bya RecQ DNA helicase family gene;

[7] the apoptosis inducer according to [6], wherein the compound thatsuppresses a function of a protein encoded by a gene belonging to theRecQ DNA helicase family is any one of:

-   (a) a mutant protein belonging to the RecQ DNA helicase family,    where the mutant protein has a dominant-negative activity towards    the protein encoded by the gene belonging to the RecQ DNA helicase    family;-   (b) an antibody that binds to the protein encoded by the gene    belonging to the RecQ DNA helicase family; or-   (c) a low-molecular-weight compound that binds to the protein    encoded by the gene belonging to the RecQ DNA helicase family;

[8] the apoptosis inducer according to any one of [1] to [7], whereinthe gene belonging to the RecQ DNA helicase is the WRN gene, the BLMgene, or the RecQ1 gene;

[9] the apoptosis inducer according to any one of [1] to [7], whereinthe nucleotide sequence of the gene belonging to the RecQ DNA helicasefamily is the nucleotide sequence of any one of SEQ ID NOs: 6 to 30;

[10] the apoptosis inducer according to any one of [2] to [4], whereineither strand of the double-stranded RNA with RNAi activity comprisesthe nucleotide sequence of any one of SEQ ID NOs: 1 to 5, 31 to 40, and43 to 69;

[11] an anticancer agent which comprises the apoptosis inducer accordingto any one of [1] to [10], as an active ingredient;

[12] a method of screening for a candidate compound for a cancer cellapoptosis inducer (a screening method for an apoptosis inducer for acancer cell), which comprises the steps of:

-   (a) contacting a test compound with a protein encoded by a gene    belonging to the RecQ DNA helicase family or a partial peptide    thereof;-   (b) assaying the binding activity between the protein, or the    partial peptide thereof, and the test compound; and-   (c) selecting a compound that binds to the protein encoded by the    gene belonging to the RecQ DNA helicase family, or the partial    peptide thereof;

[13] a method of screening for a candidate compound for a cancer cellapoptosis inducer (a screening method for an apoptosis inducer for acancer cell), which comprises the steps of:

-   (a) contacting a test compound with cells expressing a gene    belonging to the RecQ DNA helicase family or with an extract of    those cells;-   (b) determining the expression level of the gene belonging to the    RecQ DNA helicase family; and-   (c) selecting a compound that reduces the expression level by    comparing the level with that determined in the absence of the test    compound;

[14] a method of screening for a candidate compound for a cancer cellapoptosis inducer (a screening method for an apoptosis inducer for acancer cell), which comprises the steps of:

-   (a) contacting a test compound with cells or an extract of those    cells, wherein the cells have a DNA comprising a structure in which    a reporter gene is operably linked to a transcriptional regulatory    region of a gene belonging to the RecQ DNA helicase family;-   (b) determining the expression level of the reporter gene; and-   (c) selecting a compound that reduces the expression level by    comparing the level with that determined in the absence of the test    compound;

[15] a method of screening for a candidate compound for a cancer cellapoptosis inducer (a screening method for an apoptosis inducer for acancer cell), which comprises the steps of:

-   (a) contacting a test compound with a protein encoded by a gene    belonging to the RecQ DNA helicase family, or a cell expressing that    protein or an extract of that cell;-   (b) determining the activity of the protein; and-   (c) selecting a compound that reduces the activity of the protein by    comparing the level with that determined in the absence of the test    compound;

[16] the method according to any one of [12] to [15], wherein the genebelonging to the RecQ DNA helicase family is the WRN gene, BLM gene, orRecQ1 gene; and

[17] a method for producing the apoptosis inducer according to [1] or[6] as a pharmaceutical composition, which comprises the steps of:

-   (a) screening for a compound using the method according to any one    of [12] to [16]; and-   (b) combining the compound with a pharmaceutically acceptable    carrier.

Preferably, the above-mentioned genes belonging to the RecQ DNA helicasefamily are human genes belonging to the RecQ DNA helicase family.

The present inventors revealed, for the first time, that apoptosis canbe induced in cancer cells (tumor cells) by suppressing the expressionof RecQ DNA helicase family genes. Thus, the present invention providesapoptosis inducers that comprise as an active ingredient a compound thatsuppresses the expression of a RecQ DNA helicase family gene. Theapoptosis inducers of the present invention comprise the activity ofselectively inducing apoptosis in cancer cells in particular.Specifically, a preferable embodiment of the present invention providespharmaceutical agents comprising the activity of selectively(specifically) inducing apoptosis in cancer cells, where thepharmaceutical agents comprise as an active ingredient a compound thatsuppresses the expression of a RecQ DNA helicase family gene, or thefunction of the protein encoded by that gene.

The term “apoptosis” generally refers to cell death actively induced bythe cell itself due to a physiological condition. Morphological featuresof apoptosis include, for example, chromosome condensation in the cellnucleus, nuclear fragmentation, loss of microvilli on the cell surface,and cytoplasmic shrinkage. Thus, as used herein, the term“apoptosis-inducing activity” refers to, for example, the activity ofinducing in cells any of the above-described morphological features ofapoptosis, but is not limited to those described above. One skilled inthe art can appropriately assess whether apoptosis induction in cells istaking place or not.

For example, the present invention's apoptosis inducers for cancer cellscan be anticancer agents (carcinostatic agents) havingapoptosis-inducing activity as their mechanism of action.

More specifically, genes belonging to the RecQ DNA helicase family(hereinafter sometimes simply referred to as “RecQ helicase genes”)include the RecQ1 gene, WRN gene, BLM gene, RTS gene, and RecQ5 gene.Those skilled in the art can readily obtain information on thenucleotide sequences of these genes from public gene databases (forexample, GenBank). Exemplary GenBank accession numbers of the genesdescribed above are listed below.

RecQ1 gene: NM_(—)002907 (SEQ ID NO: 6), NM_(—)032941 (SEQ ID NO: 7),BC001052 (SEQ ID NO: 8), D37984 (SEQ ID NO: 9), and L36140 (SEQ ID NO:10).

WRN gene: NM_(—)000553 (SEQ ID NO: 11), AF091214 (SEQ ID NO: 12), L76937(SEQ ID NO: 13), and AL833572 (SEQ ID NO: 14).

BLM gene: U39817 (SEQ ID NO: 15), NM_(—)000057 (SEQ ID NO: 16), andBCO34480 (SEQ ID NO: 17).

RTS gene: NM_(—)004260 (SEQ ID NO: 18), AB006532 (SEQ ID NO: 19),BC020496 (SEQ ID NO: 20), and BC011602 (SEQ ID NO: 21), and BC013277(SEQ ID NO: 22).

RecQ5 gene: NM_(—)004259 (SEQ ID NO: 23), AK075084 (SEQ ID NO: 24),AB006533 (SEQ ID NO: 25), AB042825 (SEQ ID NO: 26), AB042824 (SEQ ID NO:27), AB042823 (SEQ ID NO: 28), AF135183 (SEQ ID NO: 29), and BC016911(SEQ ID NO: 30).

One skilled in the art can readily obtain amino acid sequences ofproteins encoded by the RecQ helicase genes of the present invention by,for example, using the accession numbers as indicated above. As examplesof amino acid sequences of proteins encoded by the RecQ helicase genesof the present invention, the Sequence Listing shows the amino acidsequence of the protein encoded by each of the RecQ1 gene, WRN gene, BLMgene, RTS gene, and RecQ5 gene (the amino acid sequences of RecQ1protein, WRN protein, BLM protein, RTS protein, and RecQ5 protein areshown in SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, andSEQ ID NO: 74, respectively).

Depending on the presence of polymorphic variations in the nucleotidesequences, there may be multiple accession numbers for the same RecQhelicase gene. “Polymorphism” is not restricted to single-nucleotidepolymorphisms (SNPs), such as mutations due to the substitution,deletion, or insertion of a single nucleotide, but also includesmutations due to the substitution, deletion, or insertion of severalcontinuous nucleotides. Therefore, the RecQ helicase gene nucleotidesequences are not limited to the sequences available under the accessionnumbers described above. Likewise, amino acid sequences of proteinsencoded by the RecQ helicase genes are also not limited to the aminoacid sequences shown in SEQ ID NOs: 70 to 74. Specifically, theabove-described proteins of the present invention are not limited to theamino acid sequences shown in SEQ ID NOs: 70 to 74; and include proteinsfunctionally equivalent to any one of the proteins as shown in SEQ IDNOs: 70 to 74, and comprising amino acid sequences with one or moreamino acid additions, deletions, substitutions, and/or insertions in theamino acid sequences shown in SEQ ID NOs: 70 to 74.

Preferable RecQ helicase genes of the present invention include, forexample, the WRN gene, BLM gene, and RecQ1 gene. The RecQ helicase genesof the present invention typically include, but are not limited to,those derived from animals, more preferably those derived from mammals,and most preferably those derived from humans.

Preferable compounds that suppress the expression of a RecQ helicasegene of the present invention include, for example, double-stranded RNAhaving RNA interference (RNAi) activity towards the RecQ helicase gene.In general, the term “RNAi” refers to a phenomenon where target geneexpression is inhibited by inducing disruption of the target gene mRNA.This disruption is caused by introducing into cells a double-strandedRNA that comprises, a) a sense RNA comprising a sequence homologous tothe target gene mRNA sequence, and b) an antisense RNA comprising asequence complementary to the sense RNA. While the precise RNAimechanism remains unclear, it is thought that an enzyme called DICER (amember of the RNase III nuclease family) contacts double-stranded RNA,decomposing it into small fragments called “small interfering RNA” or“siRNA”. This siRNA is also included in the double-stranded RNAcomprising RNAi activity of the present invention. Furthermore, DNAsthat allow the expression of the double-stranded RNA of the presentinvention are also included in the present invention. Specifically, thepresent invention provides DNAs (vectors) that allow the expression of adouble-stranded RNA of the present invention. These DNAs (vectors) thatallow the expression of a double-stranded RNA of the present inventionare typically DNAs comprising a structure where a DNA encoding onestrand of the double-stranded RNA, and a DNA encoding the other strandof the double-stranded RNA, are operably linked to a promoter. Thoseskilled in the art can readily prepare an above-described DNA of thepresent invention with routinely-used genetic engineering techniques.More specifically, expression vectors of the present invention can beprepared by appropriately inserting DNA encoding an RNA of the presentinvention into various known expression vectors.

The RNA used to achieve RNAi need not be completely identical(homologous) to the RecQ helicase gene or a portion of the gene; howeversuch an RNA is preferable.

The double-stranded RNA comprising RNAi activity of the presentinvention is typically a double-stranded RNA that includes a) a senseRNA comprising a sequence homologous to an arbitrary RNA region ofcontinuous nucleotide residues from the RecQ helicase gene mRNA, and b)an antisense RNA comprising a sequence complementary to the sense RNA.The above-mentioned “arbitrary RNA region of continuous nucleotideresidues” is typically 20 to 30 nucleotides in length, and preferably 21to 23 nucleotides in length. The length of the double-stranded RNA ofthe present invention is however not limited, because long-chain RNAthat has no RNAi activity when intact may decompose to form siRNAcomprising RNAi activity in cells. Furthermore, a long-chaindouble-stranded RNA corresponding to the full-length or near full-lengthmRNA of the RecQ helicase gene can be, for example, pre-digested withDICER, and the resulting decomposition products can be used as anapoptosis inducer of the present invention. The decomposition productsare expected to contain double-stranded RNA molecules (siRNA) comprisingRNAi activity. In this method, it is not necessary to select an mRNAregion expected to comprise RNAi activity. Specifically, it is notessential to determine the RecQ helicase mRNA region comprising RNAiactivity.

Usually, double-stranded RNAs comprising an overhang of severalnucleotides at their ends are known to have a high RNAi activity. It ispreferable that the double-stranded RNAs of the present inventioncomprise an overhang of several nucleotides at the ends. There is nolimitation as to the length of the overhang nucleotide sequence, but itpreferably comprises two nucleotide residues. A double-stranded RNAcomprising an overhang of, for example, TT (a thymine doublet), UU (auracil doublet), or some other nucleotide (most preferably, a moleculecomprising a double-stranded RNA of 19 nucleotides and an overhang oftwo nucleotides (TT)) can be suitably used in the present invention. Thedouble-stranded RNA of the present invention also includes molecules inwhich the overhanging nucleotides are DNAs.

Those skilled in the art can appropriately prepare the above-described“double-stranded RNA comprising RNAi activity towards a RecQ helicasegene” of the present invention based on the nucleotide sequence of aRecQ helicase gene that is the target of the double-stranded RNA. Thenucleotide sequences of RecQ helicase genes can readily be obtained frompublic gene databases, as described above. For example, adouble-stranded RNA of the present invention can be prepared based onthe nucleotide sequence of any one of SEQ ID NOs: 6 to 30. Specifically,the selection of an arbitrary consecutive RNA region of an mRNAtranscript based on the nucleotide sequence of any one of SEQ ID NOs: 6to 30, and preparation of a double-stranded RNA corresponding to thisregion would be an easy task for one skilled in the art.

It is not essential to have information on the full-length nucleotidesequence of a gene (the target gene) from which the double-stranded RNAof the present invention is derived. It is enough that the arbitrary RNAregion comprising consecutive nucleotides (for example, 20 to 30nucleotides) which is to be selected has been identified. Thus, thedouble-stranded RNA of the present invention can be prepared based onthe nucleotide sequence of a fragment of a gene, such as an ExpressedSequence Tag (EST), whose mRNA sequence has been determined partially,but not completely. The accession numbers and names of EST sequences inthe GenBank database with a high homology to human genes belonging tothe RecQ DNA helicase family are shown below. However, this listincludes only a few examples of the many EST sequences. Those skilled inthe art can readily obtain sequence information on appropriate ESTfragments from public databases.

-   (a) RecQ1 gene: BQ436743, AU130503, BI756143, BQ962215.1|BQ962215,    AU117557, BQ049370, AU129387, BQ882675, BM015357, BG392546,    AU131006, BU190089, BE870195, BM786040, BQ182943, AU138156,    AW629737, CA489724, BF031494, AA258050, AW149458, BG113470,    BG177944, AV718094, BQ952333, BQ647346, AU127897, AA830035,    BQ215072, BE708578, BM467595, AL042375, AA298927, BU858680,    CB158017, AA298835, BG536173, AU280724, BU194296, BI090858,    BG118947, AA070943, BG530720, BG529492, AW505210, AW673131,    BE888299, BE794392, CA394258, Z33439, BX093234, AA298951, AA459772,    BG542269, BF344325, BQ359629, CA396832, AA670042, BF061797,    AI652053, CB306566, CB155969, BU160790, BM263857, AW338719,    AI872607, AI560139, CA450066, CA310830, BQ776691, BM980368,    BM976615, BF436358, BE502851, AW864567, AW057629, AI685944,    AI671558, AI636512, AI478320, AI474213, AI434812, AI223294,    AA969326, AA939175, AA889357, AA804459, AA456585, AA287557,    BM151219, AI261327, AW779519, AW867647, T71941, BI559810, AW182531,    BE246521, CA450299, CA311010, and AW672788.-   (b) WRN gene: AW965099, AL707510, AL709832, BM785803, BG714322,    BM552381, BF103840, BQ305613, T39125, BG992948, BQ305407, BU633322,    CB156864, AA344201, AA373157, W40393, AA287985, AA193296, AA193286,    BM721721, R58879, AW016548, AI203498, H80461, AA835784, BQ354585,    N64051, A1025669, AW102683, BU683758, AA905633, BE939929, AI457716,    BE787985, AA778924, BG941850, and BQ774611.-   (c) BLM gene: AL556823, AL556853, BM451903, BI091601, BG199179,    BI091772, BQ230262, BG772975, BG875917, BM542461, BM040993,    BE618504, BM041661, BG574669, BI667071, BE538092, BG721596,    AW502890, BM804157, CB243435, AL120858, BX106802, BE889560,    BQ316432, BE963549, AI394601, BG397477, BG756262, BX283839,    AI097184, AW503829, AW404657, BE535950, AA747832, AI590599,    AW504704, AI114820, AA904488, BG531593, BE245802, BU588736,    BQ359304, AA974756, BE778486, BM151892, AA769336, BG192554,    BG187329, AW575595, AA214549, AA480209, AI423875, AW173139,    AI630521, CA488994, AA862803, BE940055, BU431321, AA249737,    BM150628, AW138812, AA903504, BE245666, BG210806, BG185919,    BG182955, H53763, BG217661, AA887818, AA643177-   (d) RTS gene: AL561020, BU902971, CA454998, BM557643, BU944576,    BQ065027, BQ649577, BG824628, BQ072016, AL582326, BU173357,    BU902969, BE560845, BG388102, BG337750, CA489272, BM763376,    BQ646647, BU957442, BG757074, BE514063, BU171530, BQ218075,    BE513519, BE379488, BE513709, BQ668351, BG338114, BQ883533,    BM724503, BM849415, BE295951, BI457058, BG398209, BM679729,    BM794167, BM853252, BM461584, BE466402, BE281293, BE378846,    BQ215879, BM847781, BE906701, BX116159, BE349634, BG337918, H16879,    BG339379, AW236527, BM854499, AI858255, BG954668, BF513150,    BM144460, BM144537, AI886385, AI590776, BM848938, AI765149,    CA449577, BM763607, AA768048, AA595239, BU729459, BM702983,    AI872334, AA401146, BF909603, BE790022, AA984927, BM796016,    BF935882, H16770, AI471262, AA627850, BG991513, BF800269, BX095851,    BQ576069, BG059979, BF000493, AI341050, AI206129, BE243589,    AW304768, BF115628, BE869080, AI365167, BQ651717, AA620446,    BG822953, and AL042193.-   (e) RecQ gene: BU187406, BI916256, BQ954275, BQ932656, BQ924931,    BG762566, AL525351, BQ316263, BG424888, BG721800, BQ049337,    BQ061205, BQ059196, BM449612, BM469366, BX099953, CB270726,    BG763254, BU431740, BQ375153, BF982170, AI567000, AI671940,    CB155215, AA155882, BI772198, AW368882, BQ322863, AI310229,    BF896710, BF896709, BF182856, AL600850, BI198364, BI200066,    AA155835, BE314216, BE313572, BE263134, BE261456, AI363275,    AI218469, BQ316256, AW605932, and BI116483.

Specifically, a preferred embodiment of the present invention providesan apoptosis inducer that comprises a double-stranded RNA with RNAiactivity, which comprises as one strand an RNA region of continuousnucleotides in mRNA corresponding to a gene of any one of the sequencesof SEQ ID NOs: 6 to 30 or to any of the ESTs shown above.

As described above, some identical RecQ helicase genes comprise variouspolymorphic sequences. Those skilled in the art can appropriately designa sequence of RNA expected to have RNAi activity by basing the design onany one of the above-described SEQ ID NOs: 6 to 30 or EST sequences, forexample by evaluating information on a RecQ helicase gene obtained frompublic databases for polymorphisms. An apoptosis inducer comprising suchRNA is also included in the present invention. In addition, thoseskilled in the art can also prepare an apoptosis inducer byappropriately selecting, from a number of double-stranded RNAs of thepresent invention, an RNA comprising optimal RNAi activity.

The above-described “double-stranded RNA comprising RNAi activity” ofthe present invention is preferably exemplified by the double-strandedRNAs described in FIG. 1 or 2. In addition, siRNA molecules comprising anucleotide sequence shown in FIG. 3 or the Sequence Listing as eitherstrand of the double-stranded RNA (siRNA molecules comprising thenucleotide sequence of any one of SEQ ID NOs: 1 to 5, 31 to 40, and 43to 69, and a complementary strand thereof) are also included.Specifically, an embodiment of the present invention provides cancercell-specific apoptosis inducers that comprise as an active ingredientan siRNA molecule in which either strand of the double-stranded RNAhaving an RNAi effect comprises the nucleotide sequence of any one ofSEQ ID NOs: 1 to 5, 31 to 40, and 43 to 69 (an siRNA molecule comprisingthe nucleotide sequence of any one of SEQ ID NOs: 1 to 5, 31 to 40, and43 to 69, and a complementary strand thereof).

Another preferred embodiment of the present invention provides apoptosisinducers in which the compound that suppresses gene expression of theRecQ DNA helicase family is the compound of (a) or (b) described below:

(a) an antisense nucleic acid targeting a transcript of a RecQ DNAhelicase family gene, or a portion thereof; and

(b) a nucleic acid having the ribozyme activity of specifically cleavingthe transcript of a RecQ DNA helicase family gene.

As used herein, the term “nucleic acid” refers to RNA and DNA. Methodswell known to those skilled in the art for inhibiting (suppressing) theexpression of a specific endogenous gene include those using antisensetechnology. Multiple factors contribute to the inhibition of a targetgene expression by an antisense nucleic acid. These factors include, forexample, inhibition of transcription initiation through triplexformation; inhibition of transcription through hybrid formation with asequence at the site of a local open loop structure made by RNApolymerase; inhibition of transcription through hybrid formation withthe RNA being synthesized; inhibition of splicing through hybridformation with a sequence at an intron-exon junction; inhibition ofsplicing through hybrid formation with a sequence at the site ofspliceosome formation; inhibition of transfer from the nucleus to thecytoplasm through hybrid formation with mRNA; inhibition of splicingthrough hybrid formation with a sequence at the capping site or poly(A)site; inhibition of translation initiation through hybrid formation witha sequence at the site of binding of the translation initiation factor;inhibition of translation through hybrid formation with a sequence atthe ribosome binding site near the initiation codon; inhibition ofpeptide chain elongation through hybrid formation with a sequence at thesite of the translational region or polysome binding site of the mRNA;and inhibition of gene expression through hybrid formation with asequence at the site of interaction between the protein and the nucleicacid. Thus, an antisense nucleic acid inhibits target gene expression byinhibiting various processes, such as transcription, splicing andtranslation (Hirashima and Inoue, Shin Seikagaku Jikkenkoza 2 (NewLecture for Experimental Biochemistry 2), Kakusan IV (Nucleic Acid IV),Replication and Expression of Genes; Ed., Japanese Biochemical Society,Tokyo Kagaku Dozin Co., Ltd., pp. 319-347, 1993).

Antisense nucleic acids used in the present invention may inhibit theexpression of a RecQ helicase gene through any one of the actionsdescribed above. In one embodiment, an antisense sequence is designed tobe complementary to the 5′-untranslated region of a RecQ helicase genemRNA. Thus such an antisense sequence is expected to effectively inhibittranslation of that gene. A sequence complementary to the coding regionor 3′-untranslated region can also be used for this purpose. Thus, anucleic acid comprising the antisense sequence corresponding to thesequence of the translated as well as the untranslated regions of theRecQ helicase gene can be included as an antisense nucleic acid used inthe present invention. The antisense nucleic acid to be used is ligateddownstream of an appropriate promoter and preferably ligated with asequence comprising a transcription termination signal at the 3′ end.The antisense nucleic acid to be used for clinical applications istypically a synthetic oligomer. Such synthetic oligomers include thewidely used S-oligo (phosphorothioate oligo nucleotide) in which S(sulfur) has been substituted for O (oxygen) at the phosphate esterbond, thus reducing sensitivity to nuclease digestion and maintainingantisense nucleic acid activity. S-oligo is currently being tested as anantisense drug in clinical trials where it is administered directly toaffected areas. This S-oligo is also suitable for use in the presentinvention. It is preferable that the antisense nucleic acid sequence iscomplementary to the target gene sequence or a portion thereof; howeverperfect complementarity is not necessary as long as the antisensenucleic acid effectively suppresses target gene expression. Thetranscribed RNA has preferably 90% or higher complementarity, and mostpreferably 95% or higher complementarity to the target gene transcript.The length of the antisense nucleic acid used to effectively suppresstarget gene expression is at least 15 nucleotides or longer, preferably100 nucleotides or longer, and more preferably 500 nucleotides orlonger.

The inhibition of RecQ helicase gene expression can also be achievedusing a ribozyme or ribozyme-encoding DNA. The term “ribozyme” refers toan RNA molecule comprising catalytic activity. Ribozymes can have avariety of activities, and can be designed to have the activity ofcleaving RNA in a site-specific fashion. Ribozymes such as group Iintron-type ribozymes and M1 RNA, which are RNase P ribozymes, are 400nucleotides or more in length. Others such as hammer-head and hairpinribozymes have active sites comprising about 40 nucleotides (M. Koizumiand E. Otsuka, Tanpakushitsu Kakusan Koso (Protein, Nucleic acid, andEnzyme), 1990, 35, 2191).

For example, the autolytic domain of a hammer-head ribozyme cleaves the3′ side of C15 in the sequence G13U14C15. Base pairing between U14 andA9 plays an important role in this activity, and A15 or U15 can becleaved instead of C15 (Koizumi, M. et al., FEBS Lett, 228: 228, 1988).A restriction enzyme-like RNA-cleaving ribozyme that recognizes thetarget RNA sequences UC, UU or UA can be produced by designing theribozyme such that the substrate binding site complements the RNAsequence near the target site (Koizumi, M. et al., FEBS Lett, 239: 285,1988; M. Koizumi and E. Otsuka, Tanpakushitsu Kakusan Koso (Protein,Nucleic acid, and Enzyme), 35:2191, 1990; and Koizumi, M. et al., Nucl.Acids Res., 17: 7059, 1989).

The hairpin ribozyme can also be used for the purposes of the presentinvention. This ribozyme is found, for example, in the minus strand oftobacco ring spot virus satellite RNA (Buzayan, J. M., Nature, 323: 349,1986). A target specific RNA-cleaving ribozyme can also be produced froma hairpin ribozyme (Kikuchi, Y. and Sasaki, N., Nucl. Acids Res., 19:6751, 1991; Kikuchi, H., Kagaku to Seibutsu (Chemistry and Biology), 30:112, 1992). Thus, the expression of a RecQ helicase gene of the presentinvention can be inhibited by specifically digesting the gene transcriptusing a ribozyme.

Furthermore, the present invention relates to apoptosis inducerscomprising as an active ingredient a compound that suppresses thefunction (activity) of a protein encoded by a RecQ DNA helicase familygene (herein, also abbreviated as “RecQ helicase protein”).

The RecQ helicase proteins of the present invention further includemutant and homologous proteins functionally equivalent to RecQ helicaseproteins, but whose amino acid sequences contain one or more amino aciddeletions, substitutions or additions compared to the normal RecQhelicase protein sequence. Herein, the term “functionally equivalentprotein” refers to a protein having the same activity as a RecQ helicaseprotein. Alternatively, a protein functionally equivalent to a RecQhelicase protein can be defined as a protein having, for example, 90% orhigher homology, preferably 95% or higher homology, and more preferably99% or higher homology to the amino acid sequence of the RecQ helicaseprotein.

A preferred embodiment of the present invention provides apoptosisinducers in which the compound suppressing the function (activity) of aprotein encoded by a RecQ DNA helicase gene is a compound of any one of(a) to (c) below. These compounds have the activity of inducingapoptosis in cells, cancer cells in particular, through inhibiting thefunction or activity of a RecQ helicase protein:

(a) a mutant RecQ DNA helicase family protein comprisingdominant-negative activity against a protein encoded by a RecQ DNAhelicase family gene;

(b) an antibody that binds to a protein encoded by a RecQ DNA helicasefamily gene; and

(c) a low-molecular-weight compound that binds to a protein encoded by aRecQ DNA helicase family gene.

The phrase “mutant RecQ DNA helicase protein comprisingdominant-negative activity against a protein encoded by a RecQ DNAhelicase family gene” described above in (a) refers to a mutant RecQhelicase protein comprising the function of eliminating or reducing thefunction of the endogenous wild-type protein. An example is a mutantRecQ helicase family protein reported to have no ATP hydrolysisactivity. This mutant is formed when alanine or methionine issubstituted for lysine residues in motif I (ATP-binding motif), one ofseven highly conserved helicase motifs in the helicase domain of a RecQhelicase family protein. This mutant functions as a dominant negativemutant protein. Thus, the dominant negative mutant of the presentinvention is exemplified by a mutant in which the lysine residues in theabove-described motif I have been replaced with other amino acidresidues.

The antibody that binds to a RecQ helicase protein, described in (b),can be prepared by a method known to those skilled in the art. When theantibody is a polyclonal antibody, it can be prepared, for example,using the following method: Antiserum is obtained by immunizing smallanimals such as rabbits with a natural or recombinant RecQ helicaseprotein, or a recombinant RecQ helicase protein expressed as a fusionprotein with GST in microorganisms such as E. coli, or a partial peptidethereof. The antibody is purified from the serum using, for example,ammonium sulfate precipitation, a protein A column, a protein G column,DEAE ion-exchange chromatography, an affinity column where a RecQhelicase protein or synthetic peptide thereof has been immobilized, etc.Alternatively, a monoclonal antibody can be produced by, for example,immunizing a small animal such as mouse with the RecQ helicase proteinor a partial peptide thereof, removing the spleen, gently grinding thespleen to separate cells, fusing the cells with mouse myeloma cellsusing a reagent such as polyethylene glycol, and then screening thefused cells (hybridomas) to select clones that produce antibodies thatbind to the RecQ helicase protein. These hybridomas are thentransplanted into a mouse peritoneal cavity and the ascites arecollected from the same mouse. The monoclonal antibody thus prepared canbe purified using, for example, ammonium sulfate precipitation, aprotein A column, a protein G column, DEAE ion-exchange chromatography,an affinity column where a RecQ helicase protein or synthetic peptidethereof has been immobilized, etc.

There is no limitation as to the type of antibodies of the presentinvention, as long as they can bind to a RecQ helicase protein of thepresent invention. The antibodies include human antibodies, humanizedantibodies prepared using genetic recombination, antibody fragmentsthereof, and modified antibodies, in addition to the polyclonalantibodies and monoclonal antibodies described above.

The RecQ helicase protein of the present invention to be used as animmunization antigen to obtain antibodies need not derive from the samespecies as the protein. However, mammalian proteins are preferable, forexample, proteins from mice and humans, and proteins from humans areespecially preferable.

Proteins to be used as an immunization antigen in the present inventionmay be intact proteins as well as partial peptides derived from thoseproteins. Such partial protein peptides include, for example, proteinamino (N)-terminal fragments and carboxyl (C)-terminal fragments. Asused herein, “antibody” usually refers to an antibody which reacts witha full-length protein or a fragment thereof.

In addition to obtaining the above-described hybridomas by immunizingnon-human animals with an antigen, hybridomas producing a desired humanantibody comprising binding activity with the protein can also beprepared in vitro by sensitizing human lymphocytes, for example, humanlymphocytes infected with EB virus, with the protein, cells expressingthe protein, or a lysate of those cells, and fusing these sensitizedlymphocytes with immortalized human myeloma cells, for example, U266cells. When an antibody of the present invention is intended to beadministered into human bodies (antibody therapy), a human antibody orhumanized antibody is preferable to reduce the immunogenicity.

Compounds known to bind to a protein belonging to the RecQ helicasefamily include, for example, monoclonal and polyclonal antibodiesagainst RecQ1 helicase, WRN helicase, BLM helicase, RTS helicase, andRecQ5 helicase.

Compounds of the present invention that suppress RecQ helicase geneexpression or the function (activity) of a protein encoded by that genemay be natural or synthetic compounds. Typically, the compounds can beproduced, obtained or isolated using methods known to those skilled inthe art. Such compounds include, for example, compounds comprising asingle molecule, such as organic compounds, inorganic compounds, nucleicacids, proteins, peptides, and sugars; and libraries of compounds,expression products of gene libraries, cell extracts, cell culturesupernatants, the products of fermenting microorganisms, marine organismextracts, plant extracts, and compounds purified or isolated from suchextracts.

Compounds known to inhibit the function (activity) of a RecQ helicasefamily protein include, for example, distamycin A and netropsin thathave the activity of inhibiting WRN helicase and BLM helicase (Brosh, R.M. Jr, Karow, J. K., White, E. J., Shaw, N. D., Hickson, I. D. and Bohr,V. A., “Potent inhibition of werner and bloom helicases by DNA minorgroove binding drugs.”, Nucleic Acids Res. 28, 2420-2430, 2000);3,6,9-trisubstituted acridine that has the activity of inhibiting WRNhelicase and BLM helicase (Li, J. L., Harrison, R. J., Reszka, A. P.,Brosh, R. M. Jr, Bohr, V. A., Neidle, S, and Hickson, I. D., “Inhibitionof the Bloom's and Werner's syndrome helicases by G-quadruplexinteracting ligands.”, Biochemistry 40, 15194-15202, 2001); and N-methylmesoporphyrin IX that has the activity of inhibiting E. coli RecQhelicase (Wu, X. and Maizels, N., “Substrate-specific inhibition of RecQhelicase.”, Nucleic Acids Res. 29, 1765-1771, 2001).

The present invention also provides screening methods to selectcandidate compounds for apoptosis inducers for cancer cells (screeningmethods for apoptosis inducers for cancer cells). Compounds selected bythese screening methods are expected to have apoptosis-inducingactivity.

In one embodiment of these methods, the binding of a test compound to aRecQ helicase protein or partial peptide thereof is used as an index. Acompound that binds to a RecQ helicase protein or partial peptidethereof is typically expected to have the activity of inhibiting thefunction of that RecQ helicase protein.

In the above-described method of the present invention, first, a testcompound is contacted with the protein encoded by a RecQ helicase geneor partial peptide thereof. Depending on the type of index used todetect test compound binding, the RecQ helicase protein or partialpeptide thereof can be, for example, a purified protein or partialpeptide; a protein or partial peptide expressed within or outside ofcells; or a protein or partial peptide bound to an affinity column. Thetest compounds used in this method can be labeled as required. Suchlabels include, for example, radiolabels and fluorescent labels.

The next step of this method comprises detecting the binding between thetest compound and the RecQ helicase gene protein or partial peptidethereof. The binding between the test compound and the RecQ helicaseprotein or partial peptide thereof can be detected, for example, byusing a label attached to the test compound bound to the RecQ helicaseprotein or partial peptide thereof. Alternatively, binding can bedetected by using as an index a change in RecQ helicase protein activityinduced by the binding of the test compound to that protein or partialpeptide thereof which is expressed within or outside of cells.

The next step of this method comprises selecting test compounds thatbind to the RecQ helicase gene protein or partial peptide thereof.

There is no limitation as to the type of test compound used in thepresent invention. Such compounds include, but are not limited to, forexample, single unmixed compounds of organic compounds, inorganiccompounds, nucleic acids, proteins, peptides, sugars, natural compounds,and such; or compound libraries, expression products of gene libraries,cell extracts, cell culture supernatants, products of fermentingmicroorganisms, marine organism extracts, and plant extracts; andartificially synthesized compounds.

In an alternative embodiment of the screening method of the presentinvention, first, a test compound is contacted with cells that express aRecQ DNA helicase family gene, or with a cell extract prepared from suchcells. The phrase “cells that express a RecQ DNA helicase family gene”described above include cells expressing an endogenous RecQ helicasegene, and cells into which an exogenous RecQ helicase gene has beenintroduced and in which that gene is expressed. The cells in which anexogenous RecQ helicase gene is expressed can typically be prepared byintroducing into host cells a vector which contains the gene. Thoseskilled in the art can prepare such an expression vector using routinegenetic engineering techniques. In the screening methods of the presentinvention, cells expressing a RecQ helicase gene preferably includevarious tumor cells, for example, MCF7 (breast cancer), A549 (lungcancer), U2OS (osteogenic sarcoma), C33A (cervical cancer), HT1080(fibrosarcoma), PA-1 (ovarian teratocarcinoma), Tera2 (embryonalcarcinoma), T24 (bladder cancer), K562 (chronic myelocytic leukemia),Molt4 (acute lymphoblastic leukemia), A172 (glioblastoma), HeLa(cervical cancer), and HepG2 (hepatic cancer), U251 (glioblastoma),UACC62 (melanoma), Caki-1 (renal cancer), KP4 (pancreatic cancer), MKN45(stomach cancer), and LNCaP (prostatic cancer).

Typically, but without limitation, a test compound is contacted withcells expressing a RecQ helicase gene by adding the test compound to aculture medium of the cells expressing the RecQ helicase gene. When thetest compound is a protein, the contact can be achieved by introducinginto the cells a DNA vector that allows protein expression.

The next step of this method comprises determining the expression levelof the RecQ helicase gene. Herein, the phrase “gene expression” refersto both transcription and translation. The gene expression level can bedetermined using a method known to those skilled in the art. Forexample, mRNA can be extracted from cells expressing the RecQ helicasegene according to a conventional method, and by using this mRNA as atemplate, the transcriptional level of the gene can be determined usingNorthern hybridization or RT-PCR. Alternatively, the translational levelof the gene can be determined by collecting protein fractions from thecells expressing the RecQ helicase family gene, and then detecting theexpression of the RecQ helicase protein using an electrophoresis methodsuch as sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Furthermore, the translational level of the gene can bedetermined by detecting the expression of the RecQ helicase protein byWestern blotting analysis using an antibody against the protein. Thereis no limitation as to the type of antibody used for RecQ helicaseprotein detection, as long as the protein can be detected. Suchantibodies include, for example, both monoclonal and polyclonalantibodies. For example, the above-described antibodies of the presentinvention can also be used.

In this method, a compound is then selected such that it causes areduction in expression level when compared to the expression levelmeasured in the absence of a test compound (control). The compoundselected by the above-described procedure is expected to have theactivity of inducing apoptosis in cancer cells. This compound may beused as an anticancer agent whose mode of action is based on apoptosisinduction.

In an alternative embodiment of the screening method of the presentinvention, a compound that reduces the expression level of a RecQ DNAhelicase family gene of the present invention is selected using areporter gene.

In this method, a test compound is first contacted with cells (or anextract of those cells) that comprise a DNA having a structure where areporter gene is operably linked to a transcriptional regulatory regionof a RecQ helicase gene. As used herein, the phrase “operably linked”means that the transcriptional regulatory region of a RecQ helicase geneis linked to a reporter gene in such a way as to induce reporter geneexpression when a transcriptional factor binds to the transcriptionalregulatory region of the RecQ helicase gene. Thus, even when thereporter gene is connected with another gene and thus forms a fusionprotein with that gene product, such a connection can be expressed bythe phrase “operably linked”, as long as the expression of the fusionprotein is induced when the transcriptional factor binds to thetranscriptional regulatory region of the RecQ helicase gene. Using aknown method and based on the cDNA nucleotide sequence for a RecQhelicase gene, those skilled in the art can obtain from the genome thetranscriptional regulatory region of that RecQ helicase gene.

There is no limitation as to the type of reporter gene used in thismethod, as long as it can detect the expression of the gene. Suchreporter genes include, for example, the CAT gene, lacZ gene, luciferasegene, and GFP gene. The “cells that comprise a DNA having a structurewhere a reporter gene is operably linked to a transcriptional regulatoryregion of a RecQ DNA helicase family gene” include, for example, cellsinto which a vector with the structure where a reporter gene is operablylinked to a transcriptional regulatory region of a RecQ DNA helicasefamily gene has been introduced. Those skilled in the art can preparethe above-described vector using routine genetic engineering techniques.The introduction of such a vector into cells can be achieved using aconventional method, for example, using calcium phosphate precipitation,electroporation, the lipofectamine method, microinjection, etc. “Cellsthat comprise a DNA having a structure where a reporter gene is operablylinked to a transcriptional regulatory region of a RecQ DNA helicasefamily gene” also includes cells in which that structure has beeninserted into the chromosome. The DNA structure can be inserted into thechromosome by using a method routinely used by those skilled in the art,for example, a gene transfer method using homologous recombination.

An extract of those “cells that comprise a DNA having a structure wherea reporter gene is operably linked to a transcriptional regulatoryregion of a RecQ DNA helicase family gene” includes, for example, amixture prepared by adding a DNA to a cell extract included in acommercially available in vitro transcription translation kit, wherethat added DNA comprises a structure where a reporter gene is operablylinked to a transcriptional regulatory region of a RecQ DNA helicasefamily gene.

In this method, the “contact” can be achieved by adding a test compoundinto a culture medium of “cells that comprise a DNA having a structurewhere a transcriptional regulatory region of a RecQ DNA helicase familygene is operably linked to a reporter gene”, or by adding a testcompound into the above-described commercially available cell extract,which contains the DNA. However, the method of contact is not limited tothese methods described above. When the test compound is a protein, thecontact can also be achieved, for example, by introducing into the cellsa DNA vector that directs the expression of the protein.

The next step of this method comprises determining the level of reportergene expression. The expression level of the reporter gene can bedetermined by a method that depends on the type of the reporter gene andwhich is known to those skilled in the art. For example, when thereporter gene is the CAT gene, expression level can be determined bydetecting the acetylation of chloramphenicol, mediated by the CAT geneproduct. When the reporter gene is the lacZ gene, expression level canbe determined by detecting color development in the chromogeniccompound, mediated by the catalytic action of the lacZ gene expressionproduct. When the reporter gene is the luciferase gene, the level can bedetermined by detecting the fluorescence of the fluorescent compound,mediated by the catalytic action of the luciferase gene expressionproduct. Alternatively, when the reporter gene is the GFP gene, thelevel can be determined by detecting the fluorescence of the GFPprotein.

The next step of this method comprises selecting compounds that reducereporter gene expression level as compared to expression leveldetermined in the absence of a test compound. The compounds selected bythe above-described procedure can be candidate compounds for apoptosisinducers for cancer cells.

In an alternative embodiment of the method of the present invention,compounds are screened using as an index the activity of a proteinencoded by a RecQ DNA helicase family gene of the present invention.

In this method, a test compound is first contacted with a proteinencoded by a RecQ helicase gene, or cells expressing that protein, or anextract of such cells. The activity of the protein is then determined.The activity of the protein includes, for example, DNA helicase activityand DNA-dependent ATP hydrolysis activity. Helicase activity typicallymeans the unwinding activity of double-stranded DNA into twosingle-stranded DNAs. DNA helicase activity can be determined by amethod known to those skilled in the art. For example a radio-labeledcomplementary oligo DNA is annealed with single-stranded circular M13phage DNA to form a partially double-stranded structure. Using thisstructure as a substrate, the enzyme is incubated in the presence of ATPand magnesium ions. The products of this reaction are then fractionatedinto single-stranded and double-stranded DNA using polyacrylamide gelelectrophoresis or agarose gel electrophoresis, and then detected usingautoradiography. In an alternative method, complementary single-strandedDNAs, labeled with europium and Qcy7 respectively, are annealed and theresulting double-stranded DNA is used as a substrate and incubated withthe enzyme in the presence of ATP and magnesium ions, whereupon thefluorescence of the europium-labeled single-stranded DNA released fromthe double-stranded DNA by helicase activity is determined. Alternativemethods include an electrochemiluminescence-based helicase assay,scintillation proximity assay, homogeneous time-resolved fluorescencequenching assay, and DELFIA helicase assay (Zhang, L., Schwartz, G.,O'Donnell, M. and Harrison, R. K., “Development of a novel helicaseassay using electrochemiluminescence.”, Anal. Biochem. 293, 31-37,2001).

The activity of the above-mentioned DNA-dependent ATP hydrolysis can bedetermined using a method known to those skilled in the art. Suchmethods include, for example, a method that comprises reacting theenzyme with ATP as a substrate in the presence of magnesium ions andplasmid DNA or salmon sperm DNA, and then quantifying color developmentassociated with the release of inorganic phosphoric acid in theconversion of ATP to ADP using malachite green.

A compound is then selected that reduces RecQ helicase protein activity,compared with activity determined in the absence of the test compound.In the above-described method, it is preferable to use a full-lengthRecQ helicase protein having no mutations. However, a protein whoseamino acid sequence contains partial substitutions and/or deletions canalso be used, as long as it has an activity equivalent to that of theRecQ helicase protein.

In the above-mentioned screening method, the RecQ DNA helicase familygene is preferably the WRN gene, BLM gene, or RecQ1 gene.

The present invention also provides anticancer agents (pharmaceuticalcompounds for treating cancers) which comprise as an active ingredientan apoptosis inducer of the present invention that targets cancer cells(cancer cell-specific).

The present invention also provides a method for producing an apoptosisinducer as a pharmaceutical composition. In this method a candidatecompound for the apoptosis inducer for cancer cells is first selectedusing a screening method of the present invention. Then, the selectedcompound is combined with a pharmaceutically acceptable carrier. Such apharmaceutically acceptable carrier can include, but is not limited to,for example, detergents, excipients, coloring agents, flavoring agents,preservatives, stabilizers, buffers, suspensions, isotonizing agents,binders, disintegrating agents, lubricants, fluidizing agents, andcorrectives. Other conventional carriers can be also used appropriately.

The pharmaceutical agents of the present invention can be formulated byadding the above-indicated carriers as required and according to aconventional method. More specifically, such carriers include: lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinylacetaldiethylamino acetate,polyvinylpyrrolidone, gelatin, medium chain triglyceride,polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethylcellulose, cornstarch, and inorganic salts.

The dosage forms for the agents described above include, for example,oral forms, such as tablets, powders, pills, dispersing agents,granules, fine granules, soft and hard capsules, film-coated tablets,pellets, sublingual tablets, and pastes; and parenteral forms, such asinjections, suppositories, endermic liniments, ointments, plasters, andliquids for external use. Those skilled in the art can select theoptimal dosage form depending on the administration route, subject, andsuch. Viral vectors such as retrovirus, adenovirus, and Sendai virusvectors, and non-viral vectors such as liposomes, may be used tointroduce, into the living body, DNAs expressing proteins encoded byRecQ DNA helicase family genes, or DNAs expressing antisense RNAs,ribozymes, or siRNAs that suppress RecQ DNA helicase family genes.Alternatively, non-viral vectors such as liposomes, polymer micelles, orcationic carriers, may be used to introduce, into the living body,synthetic antisense nucleic acids or synthetic siRNAs that suppress RecQDNA helicase family genes. The introduction methods include, forexample, in-vivo and ex-vivo methods.

The present invention also includes pharmaceutical compositionscomprising the above-described apoptosis-inducing activity.

Ultimately, the dose of a pharmaceutical agent or pharmaceuticalcomposition of the present invention can be appropriately determined bya physician considering the dosage form, administration method,patient's age, weight, symptoms, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleotide sequences of the siRNAs used in the Examples, and thenucleotide sequences of other siRNAs which exhibited exceedingly highgene expression suppression.

FIG. 2: A continuation of FIG. 1.

FIG. 3: The nucleotide sequences of siRNAs used in the Examples, whichwere highly capable of suppressing the expression of RecQ1, WRN, or BLMgene. “TT” represents a DNA overhang region, and the remainingnucleotides represent RNAs that are siRNAs forming double strands withthe complementary strand.

FIG. 4: A graph showing the results obtained by introducing each of thesiRNAs corresponding to the five types of human RecQ helicases, WRN,BLM, RTS, RecQ1, and RecQ5, into HeLa cells respectively, and thenquantifying each mRNA expression using Taqman PCR 72 hours later. NS isthe control RNA.

FIG. 5: Graphs showing the results obtained by semi-quantitative TaqmanPCR assays for WRN, BLM, and RecQ1 mRNA expression 72 hours after theindividual introduction of WRN, BLM, and RecQ1 siRNAs into TIG3 cells.NS is the control RNA.

FIG. 6: The result of quantification of the expression level of RecQ1,WRN, or BLM gene in HeLa cells 48 hours after introduction of siRNAagainst each gene by semi-quantitative RT-PCR. NS corresponds to thenon-silencing siRNA treatment.

FIG. 7: Photographs showing the results obtained by Western blotanalysis for the expression levels of the proteins, WRN, BLM, RTS,RecQ1, and RecQ5, in HeLa cells in which mRNA expression was revealed tobe suppressed.

In FIG. 8: Upper panel: A graph showing the results obtained byintroducing each of the five types of human RecQ helicase siRNAs intoHeLa cells, and then determining cell viability using an MTT assay fourdays later. NS is the control RNA.

Lower panel: A graph showing the results of MTT assays of cell viabilityfour days after the individual introduction of WRN, BLM, and RecQ1siRNAs into TIG3 cells. NS is the control RNA.

FIG. 9: The viability of HeLa cells determined 96 hours aftertransfection of the HeLa cells with each siRNA against RecQ1, WRN, orBLM. Shows cell counts when the number of cells transfected withnon-silencing siRNA is taken to be 100%.

FIG. 10: A graph showing the results obtained by quantifying the mRNAexpression of WRN, BLM, and RecQ1 using Taqman PCR 72 hours after theindividual introduction of WRN, BLM, and RecQ1 siRNAs into A549 cells.NS is the control RNA.

FIG. 11: Photographs showing the results obtained by Western blotanalysis for the expression of WRN, BLM, and RecQ1 proteins in A549cells in which mRNA expression was revealed to be suppressed.

FIG. 12: A graph showing the results of MTT assays of cell viabilityfour days after the individual introduction of WRN, BLM, and RecQ1siRNAs into A549 cells. NS is the control RNA.

FIG. 13: Photographs showing the results of examination by TUNEL methodof the presence of apoptosis induction 48 hours after the individualintroduction of WRN, BLM, and RecQ1 siRNAs into HeLa cells,respectively. Each left panel shows the nuclei (green) of cells in whichapoptosis was induced. Each right panel shows the nuclei of cells in thesame visual field.

FIG. 14: Photographs showing tumor growth inhibition in nude mice as aresult of the introduction of silencing RecQ1-siRNA. The photographsshow the whole sizes of the tumor injected with RecQ1-siRNA (top left)and the tumor injected with NS-siRNA (top right), 32 days afterinoculation. The bottom panel shows A549 tumors excised from the mice: atumor injected with NS-siRNA (NS) (top); and a tumor injected withRecQ1-siRNA (RecQ1) (bottom).

FIG. 15: Time-dependent changes in the inhibitory effects of WRN-siRNAand RecQ1-siRNA on the growth of A549 tumors in nude mice.

FIG. 16: Photographs showing the presence or absence of apoptosisinduction detected by TUNEL method using tumor sections prepared fromtumor (A549)-transplanted nude mice introduced with siRNA. Control,RecQ1, and WRN images were obtained by the introduction of NS siRNA,RecQ1 siRNA, and WRN siRNA, respectively. Red-brown indicates apoptoticnuclei, and blue-purple indicates non-apoptotic nuclei.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in detail below with reference toExamples, but it is not to be construed as being limited thereto.

EXAMPLE 1 Cell Culture

The human cells used were HeLa (uterocervical carcinoma cells), A549(human lung cancer cells), MCF7 (human breast cancer cells), TIG3(normal diploid fibroblast cells), U2OS (osteosarcoma), HepG2 (hepaticcancer), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (renal cancer),KP4 (pancreatic cancer), MKN45 (stomach cancer), and LNCaP (prostaticcancer). All human cells were cultured under 5% CO₂ at 37° C. inDulbecco's modified Eagle's medium containing 10% fetal bovine serum and50 μg/ml gentamycin.

EXAMPLE 2 The Effect on Cancer Cell Growth of the Suppression ofExpression of Five Types of Human RecQ Helicases

Five siRNAs targeting each of the five types of human RecQ helicasegenes (WRN, BLM, RTS, RecQ1, and RecQ5) were designed by using themethod by Elbasher et al. (Elbasher, M. S. et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494-498 (2001)) to examine the effect of suppressing theexpression of each of the five human RecQ helicases on cancer cellgrowth. The siRNAs were synthesized by Dharmacon, Inc. and QIAGEN.

FIGS. 1 and 2 show the selected siRNAs, and the sequences used tosynthesize these siRNAs (the lower-case letters represent residueshomologous to the mRNA of a RecQ DNA helicase family gene), and thesiRNA sequences that strongly suppressed gene expression, respectively.The Sequence Listing contains only sense strands sequences, and thus thecorresponding antisense strands are omitted from the Sequence Listing.

The siRNAs were introduced into human uterocervical carcinoma-derivedHeLa cells and fetal embryo-derived fibroblast TIG3 cells as normalcells. Twenty-four hours before siRNA transfection, the cells wereplated in 24-well plates at a density of 1 to 3×10⁴ cells/well. Whenthese cells were 20 to 50% confluent, the siRNAs were transfected.Twenty-four hours after plating, cells were transfected with the siRNAsusing Oligofectamine (Invitrogen) or Lipofectamine 2000 (Invitrogen)respectively, following the supplier's protocols.

Total RNA was extracted from the cells using RNeasy Mini Kits (Qiagen),42 and 72 hours after siRNA transfection, respectively. Quantitative PCRwas carried out using an ABI PRISM 7000 Sequence Detection System(Applied Biosystems). RT-PCR primers for the RecQ helicase genes andbeta-actin gene, as well as TaqMan probes were purchased from AppliedBiosystems. RT-PCR reactions were carried out using TaqMan One-StepRT-PCR Master Mix Reagents Kits (Applied Biosystems) according to themethod described in the manual. The mRNA expression levels were comparedquantitatively using beta-actin as a standard.

The expression of each mRNA in cells into which a control RNA (NS) hadbeen introduced was taken as 100%, and then compared with the expressionof each mRNA in cells into which each siRNA had been introduced.NS-siRNA is a duplex consisting of the following two strands:5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 41/“dTdT” is represented by“TT” in the Sequence Listing) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ IDNO: 42/“dTdT” is represented by “TT” in the Sequence Listing).

As a result, in HeLa cells cultured for 72 hours, the siRNAs whichreduced mRNA expression to the following values were identified: 2% inthe case of WRN; 15% in the case of BLM; 0% in the case of RTS; 0% inthe case of RecQ1; and 7% in the case of RecQ5 (FIG. 4). In contrast, inTIG3 cells, mRNA expression of WRN, BLM, and RecQ1 were suppressed downto 1%, 0%, and 9% respectively. Thus, expression was markedly suppressedwhen compared with expression in TIG3 cells in which control RNA (NS)had been introduced (FIG. 5).

Next, the present inventors also designed siRNAs against three types ofhuman RecQ helicases, RecQ1, WRN, and BLM, by the same procedure asdescribed above, and selected 14, 11, and 7 types of siRNAs for RecQ1,WRN, and BLM, respectively. The selected sequences also include somesequences already shown above. The selected siRNA sequences are shown inFIG. 3. In the Sequence Listing, only sense stands are shown whilecorresponding antisense strands are not.

The siRNAs were introduced into HeLa cells. 48 hours after introduction,the expression levels of RecQ1, WRN, and BLM genes were quantitated bysemi-quantitative RT-PCR. The gene expression levels after introductionof each siRNA were compared taking the expression levels after treatmentwith non-silencing siRNA (NS) as 100%.

As a result, all 14 types of siRNAs for RecQ1 and the siRNAs for WRN3,WRN4, WRN6, and WRN12 exhibited 90% or stronger gene-suppression effect,while the siRNAs for BLM2, BLM3, BLM4, BLM5, and BLM7 suppressed thegene expression level by 80% or more (FIG. 6).

Western blot analysis was used to determine the expression levels of theproteins WRN, BLM, RTS, RecQ1, and RecQ5 in HeLa cells in which eachmRNA expression was suppressed. The cells were harvested 48 and 72hours, respectively, after transfection with the siRNAs, and then lysedin RIPA solutions (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X 100,and 1% sodium deoxycholate). The proteins extracted were fractionatedusing SDS-PAGE on a polyacrylamide gel with a concentration gradient of4% to 20%. The fractionated proteins were transferred onto apolyvinylidene difluoride (PVDF) membrane. As the primary antibodies,polyclonal antibodies against RecQ1 and BLM, monoclonal antibodiesagainst WRN, RTS, and RecQ5, and β-actin antibody were used. Thesecondary antibody was an antibody conjugated with peroxidase. Theproteins were detected by chemical luminescence using ECL Plus WesternBlotting Detection Reagents (Amersham Biosciences).

As a result, each helicase protein was detected by using an antibodyspecific for the helicase. It was confirmed that, even at the proteinlevel, the expression of these gene products was suppressed whencompared to expression in cells into which control RNA (NS) had beenintroduced (in a 72-hour cell cultivation; FIG. 7).

EXAMPLE 3 Viability of HeLa and TIG3 Cells

Each of the five types of siRNAs against human RecQ helicases selectedin Example 2 was introduced into HeLa cells and TIG3 cells. The viablecell number after 96 hours was determined by a colorimetric assay forcell viability based on the cleavage of the tetrazolium salt WST-8 bymitochondrial dehydrogenase using Cell Count Reagent SF (NacalaiTesque). The absorbance of formazan dye at 450 nm was determined threehours after addition of the reagent.

The viability of HeLa cells in which control RNA (NS) had beenintroduced was taken as 100%. The viabilities of HeLa cells in which RTSand RecQ5 siRNAs had been introduced were 96% and 94%, respectively,showing that viability was not largely affected. However, theviabilities of HeLa cells in which WRN, BLM, and RecQ1 siRNAs had beenintroduced were 10%, 28%, and 5% respectively, indicating a markedreduction in the viability of HeLa cells (FIG. 8, upper panel). Incontrast, the viability of the resulting TIG3 cells was comparable tothat of the TIG3 cells into which NS-siRNA had been introduced. Theviabilities of cells into which WRN, BLM, and RecQ1 siRNAs were 85%,76%, and 78%, respectively. Accordingly, the introduction of WRN, BLM,and RecQ1 siRNAs caused no significant reduction in the viability ofTIG3 cells (the bottom panel in FIG. 8). These results show that thesuppression of WRN, BLM, or RecQ1 expression reduces the viability ofHeLa cells but not significantly influence the viability of TIG3 cells.

In addition, the present inventors introduced each of the newly designedsiRNAs against RecQ1, WRN, and BLM into HeLa cells, and determined thecell viability by MTT assay after 96 hours. The siRNAs used are shown inFIG. 3.

The result showed that RecQ1-9, WRN4, and BLM2 reduced the viability to20%, 5%, and 7%, respectively, when the number of cells treated withnon-silencing siRNA (NS), a control, was taken as 100% (FIG. 9).

EXAMPLE 4 Effect of siRNA on the Proliferation of A549 Cells

The effects of the three of WRN, BLM, and RecQ1 siRNAs affecting HeLacell proliferation in EXAMPLE 3, on the proliferation of human lungcancer-derived A549 cells were examined. WRN, BLM, and RecQ1 siRNAs wereintroduced into A549 cells, respectively, and 72 hours later, WRN, BLM,RecQ1 mRNA expression was quantified using Taqman PCR. The expressionlevel of each mRNA in cells in which control RNA (NS) had beenintroduced was taken as 100%. Comparison of the respective mRNAexpression levels in the cells into which each siRNA had been introducedrevealed that the mRNA expression of WRN, BLM and RecQ1 had beensuppressed to 8%, 21%, and 4% respectively (FIG. 10).

Western blot analysis was used to determine the expression levels ofWRN, BLM, and RecQ1 proteins in the A549 cells in which each mRNAexpression had been suppressed. As a result, it was confirmed that evenat the protein level the expression of these gene products wassuppressed when compared to expression in cells into which control RNA(NS) had been introduced (FIG. 11).

EXAMPLE 5 Viability of A549 Cells

Each of the siRNAs was introduced into A549 cells, respectively. Theviability of the cells after four days was assessed using an MTT assay.When the viability of the cells in which control RNA (NS) had beenintroduced was taken as 100%, the viabilities of cells into which WRN,BLM, and RecQ1 siRNAs had been introduced were 36%, 60%, and 32%respectively. As a result, the viability of A549 cells was found to bemarkedly reduced (FIG. 12).

EXAMPLE 6 Apoptosis-Inducing Activity of siRNA in HeLa Cells

Next it was investigated as to whether or not apoptosis caused theabove-described reduction in viability of the cells into which thesiRNAs had been introduced. WRN, BLM, and RecQ1 siRNAs were introducedinto HeLa cells, respectively. After 48 hours, the presence of apoptosisinduction was examined by the TUNEL method using in situ cell deathdetection kit (Roche Diagnostics) following the supplier's protocols.

Apoptosis induction was clearly recognized in HeLa cells where WRN, BLM,or RecQ1 siRNA had been introduced. However, apoptosis induction was notdetectable in HeLa cells in which control RNA (NS) had been introduced(FIG. 13). Furthermore, under the same conditions, apoptosis inductionwas also not detected in TIG3 cells.

EXAMPLE 7 Apoptosis-Inducing Activity in Various Cells

To determine whether the apoptosis-inducing activities of WRN-siRNA,BLM-siRNA, and RecQ 1-siRNA in HeLa cells are specific to the tumor cellor may occur in other tumor cell lines as well, the present inventorsevaluated the apoptosis-inducing effects of siRNAs by the same procedureas described in Example 6 using eleven types of tumor cell lines derivedfrom various tumor types: HeLa, A549 (lung cancer), MCF7 (breastcancer), HepG2 (hepatic cancer), U2OS (osteosarcoma), U251(glioblastoma), UACC62 (melanoma), Caki-1 (renal cancer), KP4(pancreatic cancer), MKN45 (stomach cancer), and LNCaP (prostaticcancer). The results showed that the siRNAs induced apoptosis in severalcancer cells but did not induce apoptosis in normal cells.

The results of investigations into the apoptosis-inducing effects of thesiRNAs tested in Examples 6 and 7 are summarized in Table 1.

TABLE 1 GENE HeLa A549 MCF7 HepG2 U2OS U251 UACC62 Caki-1 KP4 MKN45LNCaP WRN +++ ++ ++ ++ +++ +++ ++ ++ ++ ++ ++ BLM +++ ++ ++ + ++ +++ +++ ++ + ++ RecQ1 +++ ++ ++ ++ +++ ++ + ++ ++ + ++ In the Table above,“+++”, “++”, and “+” indicate that apoptosis was induced in 70% or more,70% to 40%, and 40% to 20% of cells, respectively.

All eleven types of tumor cell lines listed above were sensitive to thesiRNAs against the three types of RecQ helicases, although the level ofsensitivity varied depending on the siRNA combination and tumor cellline. When considering transfection efficiency, the effect of each siRNAon tumor cells should be more strictly evaluated. However, what deservesemphasis is that WRN-siRNA, BLM-siRNA, and RecQ1-siRNA are effective fora broad range of tumor types and thus can be candidates for excellentanti-tumor agents. In addition, WRN, BLM, and RecQ1 helicases arepromising as tools in screenings for anti-tumor agents.

EXAMPLE 8 Inhibition of Tumor Cell Growth in Tumor-Bearing Animal Modelsby siRNAs

The present inventors tested whether the RecQ helicase siRNA-mediatedselective inhibition of tumor cell growth found in vitro was alsoachieved in vivo in cancer-bearing animal models.

Male BALB/cA nude mice were purchased from CLEA Japan, Inc. A549 cells(5×10⁶ cells/0.1 ml) were subcutaneously injected into the back of thenude mice (six- to seven-week-old). siRNA administration was commencedeight days after subcutaneous injection of the tumor cells.

WRN-siRNA and RecQ1-siRNA were tested for their tumor-suppression effecton the A549 tumor which had been grown in the backs of nude mice (FIGS.14 and 15). 25 μg of 5′-phosphorylated siRNA was combined with 5 μg ofpolyethylene imine (molecule weight: 10,000; Wako) in 50 μl ofphysiological saline. The resulting mixture was injected every threedays (day 8, 11, 14, 17, 20, 23, 27, and 32) into the top of the solidtumors (tumor volume: approximately 40 mm³) grown in the eight daysafter A549 cell inoculation. The tumor volumes were determined using avernier caliper. The formula for estimating the volumes of theellipsoidal tumors was: L×W²/2 (wherein, L represents the major axis oftumors and W represents the minor axis). The tumor volumes were analyzedfor statistical significance by a multiple comparison test (Holm-Sidaktest).

The result showed that the development of the A549 tumor was markedlysuppressed over 32 days after inoculation of RecQ1-siRNA. However,NS-siRNA mixed with polyethylene imine by the same procedure asdescribed above did not have any effect and caused the A549 tumor toincrease in volume (FIG. 14). No weight loss was observed in the mice towhich the mixture of RecQ1-siRNA and polyethylene imine was injected ascompared to the untreated, cancer-bearing mice. This indicates that thistreatment has no serious adverse effect.

In view of these results, the in vivo efficacy of another siRNA,specifically WRN-siRNA, was evaluated in addition to RecQ1-siRNA usingmore mice (N=6 for testing each siRNA) by periodically measuring tumorvolume for 50 days and plotting the mean tumor volume. WRN-siRNA andRecQ1-siRNA were introduced together with a polyethylene imine carrierinto each subject by the same procedure as described in the figurelegend for FIG. 14. A suspension of polyethylene imine and NS-siRNAprepared by the same procedure as described above was used as a controlto evaluate the adverse effects of the pharmaceutical carrier and siRNAthemselves.

FIG. 15 shows that, by day 50, RecQ1-siRNA had efficiently inhibited theincrease in tumor volume by about 80% (P<0.01) while WRN-siRNA inhibitedit by about 50% (P<0.05) as compared to NS-siRNA (PEI/control).

Furthermore, it was assessed whether or not apoptosis was induced inRecQ1-siRNA- or WRN-siRNA-introduced solid tumor tissues. Tumor tissueswere excised 23 days after introduction of RecQ1-siRNA or WRN-siRNA, andfixed with a 4% paraformaldehyde fixative. Then, the tissues wererefixed with a neutralized 10% formalin buffer fixative. After afive-day fixation, the tissues were sufficiently washed with water andembedded in paraffin using an automated embedding system. Then, paraffinsections of about 2 μm were prepared, and apoptosis was detected usingthe ApopTag Peroxidase in situ Apotosis Detection Kit (SerologicalsCo.). The result showed that apoptosis-positive cells were found at asignificant level in solid tumor tissues to which RecQ1-siRNA orWRN-siRNA had been introduced, as compared to NS-siRNA (FIG. 16).

INDUSTRIAL APPLICABILITY

Compounds that inhibit the expression of a RecQ DNA helicase family geneof the present invention or the function of a protein encoded by thegene, have the activity of selectively inducing apoptosis in cancercells. Pharmaceutical agents comprising such a compound can be used withvery few side effects as anticancer agents whose mechanism is based onthis apoptosis-inducing activity. The present invention, for the firsttime, provides anticancer agents which target RecQ helicase using amechanism based on apoptosis-inducing activity.

Even if certain compounds are found to have apoptosis-inducing activity,it is difficult to use the compounds as pharmaceuticals when theirapoptosis-inducing activities in normal cells are unknown. This isbecause there may be a risk of adverse effects when the compounds haveapoptosis-inducing activities in normal cells. In other words, if thecompounds have the apoptosis-inducing activities not specific to cancercells, in general, it is practically difficult to use the compounds aspharmaceuticals. Accordingly, the pharmaceutical agents (the compounds)of the present invention are very practical and effective because theirapoptosis-inducing activities are specific to cancer cells.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A method for inducing apoptosis of cancercells comprising a step of administering a double-stranded RNA that hasRNAi activity towards a WRN gene and suppresses the expression of theWRN gene, into a subject, wherein the double-stranded RNA inducesapoptosis in cancer cells but does not induce apoptosis in normal cellsand comprises a sense RNA comprising a sequence homologous to anarbitrary 20 to 30 consecutive nucleotides from the mRNA of the WRNgene, and an antisense RNA, comprising the sequence complementary to thesense RNA, and wherein either strand of the double-strand RNA with RNAiactivity comprises the nucleotide sequence of any one of SEQ ID NOs: 2,35, 36, and 53-62.